MicroRNA-425-5p promotes breast cancer cell growth by inducing PI3K/AKT signaling

Abstract

MicroRNA-425-5p (miR-425-5p) has been reported to be involved in the tumorigenesis of several tumors, but its function in breast cancer is still unknown. In this study, miR-425-5p was found significantly upregulated in breast cancer cells, and predicted a poor prognosis for breast cancer patients. Overexpression of miR-425-5p could significantly promote breast cancer cell growth. Further studies showed that overexpression of miR-425-5p upregulated the protein levels of Cyclin D1, Cyclin D3, CDK4, and CDK6. However, inhibiting miR-425-5p downregulated their expression and induced cell cycle arrest at G0/G1 phase. In mechanism, overexpression of miR-425-5p increased the phosphorylation of PI3K p85 and AKT, but inhibiting miR-425-5p displayed opposite effects. Moreover, miR-425-5p bound to the 3'UTR of PTEN mRNA, and downregulated the expression levels of PTEN in both mRNA and protein levels in breast cancer cells. Collectively, the results above demonstrated that miR-425-5p was involved in the tumorigenesis of breast cancer by inducing PI3K/AKT signaling and indicated that miR-425-5p could be as a potential target for breast cancer therapy in the future.

INTRODUCTION

Breast cancer is a solid malignancy occurring in the epithelial tissue of breast. Among these, 99% of breast cancer occurs in women, but only 1% in men. The incidence of breast cancer in the world has been on the rise since 1970s. In the United States, one in eight women will develop breast cancer in their life time. And in China, the growth rate of breast cancer incidence is 1% to 2% points higher than that in high-incidence countries in recent years. Thus, breast cancer has become a major public health problem in the current society. Although significant progress has been done in the treatment of breast cancer, and the diagnosis and therapeutic advances have improved the survival of breast cancer patients with early stages, some patients become resistant and some patients with late stages are still untreatable.

MicroRNA (miRNA) is a class of non-coding single-stranded RNA molecules with 22 nucleotides encoded by endogenous genes. They participate in the regulation of post-transcriptional gene expression in plants and animals. It is known that several miRNAs can also regulate the same gene, and it is presumed that miRNAs regulate one third of human genes. And recent evidences suggest that mutations or abnormal expression of miRNAs are associated with a variety of human cancers, and miRNAs can function as tumor suppressor genes or oncogenes. Studies have shown that miRNAs can inhibit the expression of important tumor-related genes and may play an important role in the diagnosis and treatment of cancer. Thus, targeting miRNAs has been as a promising approach for cancer diagnosis or treatment in clinic.

In this study, microRNA-425-5p (miR-425-5p) was found elevated in breast cancer cell lines and tumor tissues, and predicted a poor prognosis for breast cancer patients. miR-425-5p could markedly promote breast cancer cell growth by inducing PI3K/AKT signaling. This study indicated that miR-425-5p could be as a potential biomarker for breast cancer diagnosis or treatment in the future.

MATERIALS AND METHODS Cells, tissues, and chemicals

Breast cancer cell lines and the normal breast cell line MCF-10A were purchased from American Type Culture Collection (Manassas). All cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. The primary breast cancer tissues and individual normal para-cancerous tissues were collected from the First Affiliated Hospital of Soochow University. The collection and use of human tissues for this study were approved by the Institutional Review Board of the First Affiliated Hospital of Soochow University. And informed consent was received from the participants. LY294002 was purchased from Selleck Chemicals (Houston, Texas).

Quantitative real-time polymerase chain reaction (qRT-PCR)

To determine the mRNA levels of PTEN, qRT-PCR was performed using SYBR Green qPCR Master Mix (Clontech Laboratories, Inc.). The primers used were as follows: PTEN, forward 5′-ACCATAACCCACCACAGC-3′ and reverse 5′-ACCAGTTCGTCCCTTTCC-3′; GAPDH, forward 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse 5′-TGGTGAAGACGCCAGTGGA-3′.

To determine the miRNA levels of miR-425-5p, the qRT-PCR was performed as described previously. Bulge-loop miRNA qRT-PCR Primer Sets specific for miR-425-5p were designed by RiboBio (Guangzhou, China). The total RNA was extracted using a MiRNeasy Mini Kit (QIAGEN, Hilden, Germany), and the miRNA bulge-loop was reverse transcribed with the Quantscript RT Kit (QIAGEN). The relative amount of miRNAs was normalized against U6 snRNA.

Cell growth and viability

Viable cells of breast cancer cells were measured by Cell Counting Kit-8 (CCK-8) assay according to the manufacturer's instructions (Dojindo, Japan).

Immunoblotting

Immunoblotting was performed as described previously. The primary antibodies Cyclin D1, Cyclin D3, CDK4, CDK6, p-PI3K p85 (Tyr458), PI3K p85, p-AKT (Ser473), AKT, PTEN, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA). Anti-mouse and anti-rabbit immunoglobulin G (IgG) horseradish peroxidase conjugated antibodies were purchased from Beyotime Biotechnology (Nantong, China).

Cell cycle analysis

The cell cycle analysis was performed as described previously. MCF7 cells were transfected with miR-NC or miR-425-5p inhibitor for 72 hours before cell cycle analysis. Subsequently, cells were fixed and washed with cold PBS, followed by RNaseA treatment. Then, cells were washed with cold PBS and incubated with propidium iodide (PI) for 5 minutes, followed by cell cycle analysis on a flow cytometer (FACSCaliburTM; Becton Dickinson).

miRNA construction and transfection

The miR-425-5p mimic, inhibitor and a negative control (miR-NC) were synthesized from Guangzhou Ribobio Co., Ltd (Guangzhou, China). MCF7 and MDA-MB-453 cells were transfected with miR-NC, miR-425-5p mimic or inhibitor by Lipofectamine RNAiMAX Reagent (Invitrogen) in Opti-MEM medium (Invitrogen) according to the manufacturer's instruction.

Luciferase assay

The fragments containing predicted miR-425-5p binding sites or the mutants were amplified and subcloned into pGL3 vector. Subsequently, these plasmids along with miR-NC or miR-425-5p were transfected into MCF7 cells using Lipofectamine2000 (Invitrogen). Forty-eight hours later, the cells were prepared for luciferase assay using the Dual-Luciferase Reporter Assay System (Promega, Madison, Wisconsin) according to the manufacturer's instruction.

Statistical analysis

The student's t test was used for comparisons of two groups in the experiments, and all statistical tests were two-sided. A P value <.05 was considered statistically significant in this study.

RESULTS miR-425-5p is upregulated and promotes cell growth in breast cancer

To evaluate the expression levels of miR-425-5p in breast cancer, 20 pairs of breast cancer samples were collected for qRT-PCR analysis. As shown in Figure A, miR-425-5p was markedly upregulated in breast cancer tissues compared with the normal. Subsequently, a panel of breast cancer cell lines and a normal breast cell line were also collected, and the qRT-PCR showed that miR-425-5p was also elevated in various breast cancer cell lines (Figure B). In addition, the KM plotter based on the breast cancer miRNA database indicated that miR-425 predicted a poor prognosis in breast cancer (Figure C). The upregulation of miR-425-5p in breast cancer prompted us to investigate whether miR-425-5p regulated cell growth of breast cancer. As shown in Figure D-F, overexpression of miR-425-5p significantly promoted breast cancer cell growth in both of MCF7 and MDA-MB-453 cells.

miR-425-5p is upregulated and promotes cell growth in breast cancer. A, Twenty pairs of breast cancer samples were prepared for quantitative real-time polymerase chain reaction (qRT-PCR) to measure miR-425-5p expression. U6 was used as an internal control. B, Indicated cell lines were prepared for qRT-PCR to measure miR-425-5p expression. C, The survival periods of breast cancer patients with low or high miR-425 expression were estimated by Kaplan–Meier plotter (http://kmplot.com). D & E, miR-NC and miR-425-5p mimic were transfected into MCF7 (C) or MDA-MB-453 (D) cells for indicated time, followed by CCK-8 assay. F, MCF7 and MDA-MB-453 cells were transfected with miR-NC or miR-425-5p mimic respectively. Three days later, cells were prepared for qRT-PCR to detect miR-425-5p expression. *P [less than] .05; **P [less than] .01

miR-425-5p regulates cell cycle progression in breast cancer cells

As is known to all, cell growth is controlled by cell cycle or cell apoptosis. Then, some cell cycle-related proteins were examined. As shown in Figure A,B, overexpression of miR-425-5p markedly upregulated the expression levels of Cyclin D1, Cyclin D3, CDK4 and CDK6, which were all involved in the transition from the G1 to S phase of cell cycle. In contrast, inhibition of miR-425-5p by miR-425-5p inhibitor significantly reduced the expression levels of Cyclin D1, Cyclin D3, CDK4, and CDK6 (Figure C,D). And the flow cytometer analysis also showed that inhibition of miR-425-5p could induce cell cycle arrest at G0/G1 phase (Figure E). Therefore, we can conclude that miR-425-5p mediates cell growth by regulating cell cycle progression in breast cancer cells.

miR-425-5p regulates cell cycle progression in breast cancer cells. A & B, miR-NC and miR-425-5p mimic were transfected into MCF7 or MDA-MB-453 cells for 72 hours, followed by immunoblotting against Cyclin D1, Cyclin D3 (A), CDK4 and CDK6 (B). GAPDH was used as a loading control. C & D, MCF7 and MDA-MB-453 cells were transfected with miR-NC or miR-425-5p inhibitor for 72 hours, followed by immunoblotting against Cyclin D1, Cyclin D3 (C), CDK4 and CDK6 (D). E, MCF7 cells were transfected with miR-NC or miR-425-5p inhibitor for 72 hours, followed by cell cycle analysis using PI on a flow cytometer

miR-425-5p regulates PI3K/AKT signaling in breast cancer cells

Further studies revealed that miR-425-5p could regulate PI3K/AKT signaling in breast cancer cells. As shown in Figure A, overexpression of miR-425-5p by miRNA mimics could significantly increase the phosphorylation of PI3K p85 and AKT, both of which were the important regulators in PI3K/AKT signaling, in MCF7 and MDA-MB-453 cells. In contrast, inhibition of miR-425-5p by miRNA inhibitors obviously decreased the activation of PI3K p85 and AKT in both of MCF7 and MDA-MB-453 cells (Figure B). To further confirm it, a PI3K inhibitor LY294002 was added, and the results showed that the PI3K inhibitor LY294002 could significantly reduce miR-425-5p-induced cell growth progression in MCF7 cells (Figure C). And LY294002 enhanced the cell growth inhibition mediated by miR-425-5p inhibitor in MCF7 cells (Figure D). These results showed that miR-425-5p mediated PI3K/AKT signaling in breast cancer cells.

miR-425-5p regulates PI3K/AKT signaling in breast cancer cells. A, MCF7 and MDA-MB-453 cells were transfected with miR-NC or miR-425-5p mimic for 3 days, followed by immunoblotting against p-PI3K p85, PI3K p85, p-AKT, and AKT. B, MCF7 and MDA-MB-453 cells were transfected with miR-NC or miR-425-5p inhibitor for 3 days, followed by immunoblotting against p-PI3K p85, PI3K p85, p-AKT, and AKT. C, MCF7 cells were transfected with miR-NC or miR-425-5p mimic for 24 hours, and then cells were treated with vehicle or 5 μM LY294002 for 24 hours, followed by CCK-8 assay. D, MCF7 cells were transfected with miR-NC or miR-425-5p inhibitor. Twenty-four hours later, cells were treated with vehicle or 5 μM LY294002 for 24 hours, followed by CCK-8 assay. **P [less than] .01

miR-425-5p inhibits PTEN expression in breast cancer cells

Subsequently, miR-425-5p was predicted to bind to the 3'UTR of PTEN mRNA by TargetScanHuman 7.2 database (Figure A). As is known to all, PTEN was a negative regulator of PI3K/AKT signaling, and inhibiting the expression levels of PTEN could promote PI3K/AKT signaling, which was consistent with our hypothesis in this study. To confirm this prediction, luciferase assay was performed. As shown in Figure B, miR-425-5p significantly downregulated wild-type PTEN 3'UTR-driven luciferase activity, but miR-425-5p displayed no significant effect on mutant PTEN 3'UTR-driven luciferase activity, which further indicated that miR-425-5p targeted PTEN. To further evaluate the mechanism of miR-425-5p on PTEN, qRT-PCR, and immunoblotting were carried out and the results showed that overexpression of miR-425-5p markedly inhibited the mRNA level and protein level of PTEN in breast cancer cells (Figure C,D).

miR-425-5p inhibits PTEN expression in breast cancer cells. A, The predicted binding sites of miR-425-5p in the 3'UTR of PTEN mRNA by TargetScanHuman 7.2 database (http://www.targetscan.org/vert_72/). B, miR-NC or miR-425-5p, along with wild-type (WT) or mutant (Mut.) PTEN 3'UTR were co-transfected into MCF7 cells for 48 hours, followed by luciferase assay. C & D, MCF7 and MDA-MB-453 cells were transfected with miR-NC or miR-425-5p mimic for 72 hours, followed by quantitative real-time polymerase chain reaction (qRT-PCR) (C) or immunoblotting (D) to detect PTEN expression. **P [less than] .01. n.s., non-sense

DISCUSSION

miR-425-5p has been reported to be involved in the tumorigenesis of several tumors. In gastric cancer, miR-425-5p was overexpressed in tumor tissues, and its overexpression may be associated with depth of invasion and TNM stages and could be as a marker of poor prognosis. In addition, inhibiting miR-425-5p could induce cell cycle arrest at G0/G1 phase, and promote cell apoptosis in gastric cancer. Another paper reported that miR-425-5p may contribute to gastric cancer progression through a mechanism involving CYLD. CYLD was a direct target of miR-425-5p, and miR-425-5p inhibited CYLD expression in gastric cancer. In hepatocellular carcinoma (HCC), miR-425-5p was found elevated and correlated with poor prognostic clinicopathological features and low post-operative long-term survival. And SCAI and PTEN were determined to be downstream targets of miR-425-5p in HCC. miR-425-5p was also reported to be a potential prognostic biomarker for cervical cancer. Moreover, miR-425-5p was involved in regulating chemoresistance in colorectal cancer cells. It showed that miR-425-5p was markedly upregulated in HCT116-R compared with parental HCT116 cells, and inhibiting miR-425-5p reversed chemoresistance in HCT116-R cells. PDCD10 was found to be the direct target of miR-425-5p that was required for the regulatory role of miR-425-5p in chemoresistance. In this study, miR-425-5p was found upregulated in breast cancer, and predicted a poor prognosis for breast cancer patients. Overexpression of miR-425-5p significantly promoted breast cancer cell growth, and miR-425-5p regulated cell cycle progression of breast cancer cells, which suggested that miR-425-5p was a potential biomarker for breast cancer.

PI3K is a lipid kinase which induces cell cycle progression, cell survival, and cell migration; however, PTEN can reverse this process, and is a negative regulator of PI3K/AKT signaling. Many evidences have shown that PI3K/AKT signaling is constitutively activated in many tumors with PTEN dysfunction. In this paper, our studies showed that miR-425-5p regulated PI3K/AKT signaling by targeting PTEN in breast cancer cells. And the proposed model for miR-425-5p in breast cancer cells has been shown in Figure .

A proposed model for miR-425-5p in breast cancer cells. In breast cancer cells, miR-425-5p bound to the 3'UTR of PTEN mRNA, and inhibited the expression of PTEN, which resulted in inducing PI3K/AKT signaling and promoted cell growth of breast cancer

CONCLUSION

In conclusion, our present study demonstrated that miR-425-5p exerted its oncogenic effect by inducing PI3K/AKT signalling in breast cancer cells. This study suggested that miR-425-5p could be as a potential target for breast cancer treatment in the future.

CONFLICT OF INTEREST

All authors declare no conflict of interest.